Nodal Signaling Regulates the Bone Morphogenic Protein Pluripotency Pathway in Mouse Embryonic Stem Cells

Emily D. Travis,Jay L. Vivian,Della Yee,Terry Magnuson,Katherine E. Galvin

doi:10.1074/jbc.m109.077347

Abstract

Members of the transforming growth factor-beta superfamily play essential roles in both the pluripotency and differentiation of embryonic stem (ES) cells. Although bone morphogenic proteins (BMPs) maintain pluripotency of undifferentiated mouse ES cells, the role of autocrine Nodal signaling is less clear. Pharmacological, molecular, and genetic methods were used to further understand the roles and potential interactions of these pathways. Treatment of undifferentiated ES cells with SB431542, a pharmacological inhibitor of Smad2 signaling, resulted in a rapid reduction of phosphorylated Smad2 and altered the expression of several putative downstream targets. Unexpectedly, inhibition of the Nodal signaling pathway resulted in enhanced BMP signaling, as assessed by Smad1/5 phosphorylation. SB431542-treated cells also demonstrated significant induction of the Id genes, which are known direct targets of BMP signaling and important factors in ES cell pluripotency. Inhibition of BMP signaling decreased the SB431542-mediated phosphorylation of Smad1/5 and induction of Id genes, suggesting that BMP signaling is necessary for some Smad2-mediated activity. Because Smad7, a known inhibitory factor to both Nodal and BMP signaling, was down-regulated following inhibition of Nodal-Smad2 signaling, the contribution of Smad7 to the cross-talk between the transforming growth factor-beta pathways in ES cells was examined. Biochemical manipulation of Smad7 expression, through shRNA knockdown or inducible gene expression, significantly reduced the SB431542-mediated phosphorylation of Smad1/5 and induction of the Id genes. We conclude that autocrine Nodal signaling in undifferentiated mouse ES cells modulates the vital pluripotency pathway of BMP signaling.

Highlights

That the germ layers arise to produce all cell types of the adult
SB431542 inhibits ALK4/5/7 receptor signaling as a competitive inhibitor of ATP binding to the kinase domain and has no effect on bone morphogenic proteins (BMPs)-regulated Activin receptor-like kinases (ALKs) or other kinase pathways [30, 31]
In non-manipulated embryonic stem (ES) cells grown in serum-free media, substantial pSmad2 was observed via Western analysis (Fig. 1A), demonstrating an autocrine signaling activity for this pathway

Summary

Introduction

That the germ layers arise to produce all cell types of the adult. The capacity of ES cells to either self-renew or differentiate into cells of the three germ layers provides an excellent tool for studying early embryonic development, including self-renewal and pluripotency [1]. Smad2 Signaling Regulates Transcription of Multiple Feedback and Pluripotency Genes—To determine the target genes of Smad2 signaling in undifferentiated ES cells, changes in gene expression were examined following treatment with recombinant Activin or SB431542 under defined media culture conditions [20].

Results

Conclusion